A tobacco callus strain, OMT-53, was selected from many cultures as a desirable strain having high nicotine producing capacity. Several culture conditions were examined, aiming to get higher nicotine production with the callus strain, OMT-53. It was revealed that the nicotine production was remarkably enhanced when the callus tissues were cultured at a limited concentration of α-NAA in culture medium. The optimal concentrations of sucrose and nitrogen in the culture medium were 3% and 840mgN/L respectively. Some precursors in nicotine biosynthesis were examined, and only ornithine gave a slightly positive effect at 2×10^-4M concentration. Cultures at 25°C produced the highest yield for nicotine. Con-siderable amounts of nicotine (ca. 20% of total nicotine) were also recognized in the culture medium. Under the best culture condition mentioned above, nicotine production in tobacco callus tissues has been elevated to 2.14% on D.W. basis at 4 weeks' culture. This value is near to that of the intact tobacco plants.

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